An active, cell-free system from Saccharomyces cerevisiae has been developed, that translates exogenous mRNA templates accurately. The preparations aminoacyl-tRNA synthetases, tRNAs, ribosomal subunits, ribosomes, and protein factors required for translation. A strategy and procedures have also been developed that allow the quantitative analysis of intermediary reactions in the initiation, elongation and termination of protein synthesis. Three temperature-sensitive mutants of yeast are available that are altered in protein biosynthesis components. In the intact cell, two of the mutants accumulate ribosomal subunits, while polysomes are disaggregated at the non-permissive temperature. This suggests that the temperature-sensitive component(s) is involved in initiation; other analyses indicate that the mutations are in different complementation groups. The third ts mutant shows that polysomes remain aggregated at the non-permissive temperature, suggesting that an elongation component is altered. Using the approaches developed in this laboratory, the temperature-sensitive reactions and the components involved will be identified, isolated and characterized. Studies on the structural and functional characterization of two putative initiation factors from rat liver will be continued. One of these is a protein that catalyzes the specific binding of initiator tRNA to 40S ribosomal subunits in the presence of template, but does not require GTP. The other protein carries out dissociation of 80S ribosomes into subunits, and prevents the reassociation of subunits to form ribosomes, but in contrast to eIF-3 which has a very high molecular weight (500,000 - 700,000 daltons), this factor has a relatively low molecular weight, near 100,000.